Transferring the concept of multinuclearity to ruthenium complexes for improvement of anticancer activity

Article abstract of DOI:10.1021/jm8013234

Multinuclear platinum anticancer complexes are a proven option to overcome resistance of established anticancer compounds. Transferring this concept to ruthenium complexes led to the synthesis of dinuclear Ru(II)-arene compounds containing a bis(pyridinon

Full text of DOI:10.1021/jm8013234

916
J. Med. Chem. 2009, 52, 916–925
Articles
Transferring the Concept of Multinuclearity to Ruthenium Complexes for Improvement of
Anticancer Activity
Maria G. Mendoza-Ferri,† Christian G. Hartinger,*,†,‡ Marco A. Mendoza,§,| Michael Groessl,† Alexander E. Egger,†
Rene E. Eichinger,† John B. Mangrum, Nicholas P. Farrell, Magdalena Maruszak,# Patrick J. Bednarski,# Franz Klein,§
Michael A. Jakupec,† Alexey A. Nazarov,*,†,‡ Kay Severin,‡ and Bernhard K. Keppler†
UniVersity of Vienna, Institute of Inorganic Chemistry, Austria, Institut des Sciences et Inge´nierie Chimiques, Ecole Polytechnique Fe´de´rale de
Lausanne, Switzerland, Department of Chromosome Biology, Max F. Perutz Laboratories, UniVersity of Vienna, Austria, Department of
Chemistry, Virginia Commonwealth UniVersity, Richmond, Virginia, and Department of Pharmaceutical and Medicinal Chemistry,
UniVersity of Greifswald, Germany
ReceiVed October 20, 2008
Multinuclear platinum anticancer complexes are a proven option to overcome resistance of established
anticancer compounds. Transferring this concept to ruthenium complexes led to the synthesis of dinuclear
Ru(II)-arene compounds containing a bis(pyridinone)alkane ligand linker. A pronounced influence of the
spacer length on the in vitro anticancer activity was found, which is correlated to the lipophilicity of the
complexes. IC50 values in the same dimension as for established platinum drugs were found in human tumor
cell lines. No cross-resistance to oxoplatin, a cisplatin prodrug, was observed for the most active complex
in three resistant cell lines; in fact, a 10-fold reversal of sensitivity in two of the oxoplatin-resistant lines
was found. (Bio)analytical characterization of the representative examples showed that the ruthenium
complexes hydrolyze rapidly, forming predominantly diaqua species that exhibit affinity toward transferrin
and DNA, indicating that both proteins and nucleobases are potential targets.
Introduction
With the objective of developing compounds with a new
mode of action in comparison to the established anticancer drugs
cisplatin, transplatin, and oxaliplatin for treatment of a broader
range of tumors and with fewer side effects, many metal
complexes were investigated in recent years for their tumor-
inhibiting properties.1 In order to achieve a modified mechanism
of activity, several approaches were considered promising; e.g.,
the exchange of platinum centers by other metals,2,3 the
connection of more than one platinum center,1,2 platinum
complexes with trans geometry,2 and conjugation of metallo-
drugs to proteins or other biomolecules as targeting concepts.2,4-6
Figure 1. Structures of BBR3464 and organometallic Ru(II)-arene
In BBR3464 two of these concepts are realized within one
complexes of interest as anticancer agents.
compound (Figure 1); i.e., three trans-configured Pt moieties
orders of magnitude higher than that of cisplatin.8 Recently,
are linked by diaminoalkanes, and the compound shows a much
higher degree of DNA interstrand cross-linking than cisplatin.7
the compound finished a phase I clinical trial in which
preliminary activity against pancreatic, non-small-cell lung, and
ovarian cancer as well as melanoma was shown.9-11 However,
in subsequent phase II studies no major responses were observed
in small cell lung cancer as well as gastric and gastroesphageal
adenocarcinoma patients when BBR3464 was applied as a single
agent.2,9,11
Another concept for improving the chemotherapeutic activity
was realized by replacing the platinum center with ruthenium.
So far only two Ru(III) coordination compounds have reached
clinical trials, NAMI-A {H2Im[trans-RuCl4(DMSO)(Him)] (HIm
) imidazole)} and KP1019 {H2Ind[trans-RuCl4(HInd)2] (HInd
) indazole)}, and both of them are considered promising drug
candidates.12,13 Binding to plasma proteins14-21 and the reduc-
tion of Ru(III) to Ru(II)22,23 seem to be key steps in their mode
of action and the thereby-mediated degree of selectivity is
regarded as a reason for their low general toxicity.24,25 More
Furthermore, a 4-fold positively charged Pt complex was not
known before to have antineoplastic activity. The high in vitro
activity was also demonstrated for BBR3464 in cisplatin-
resistant cell lines; in fact, the activity was found to be 2-3
* To whom correspondence should be addressed. For C.G.H.: (address)
Institute of Inorganic Chemistry, University of Vienna, Waehringer Strasse
42, A-1090 Vienna, Austria; C.G.H. and A.A.N.: (phone) +43 1 4277
52600; (fax) +43 1 4277 9526; C.G.H. (e-mail) christian.hartinger@
univie.ac.at; A.A.N. (e-mail) alex.nazarov@univie.ac.at.
†
University of Vienna.
Ecole Polytechnique Fe´de´rale de Lausanne.
University of Vienna.
Current address: Institut de Ge´ne´tique et de Biologie Mole´culaire et
‡
§
|
Cellulaire, CNRS, INSERM, ULP, Colle`ge de France, 67404 Illkirch,
France.
Virginia Commonwealth University.
University of Greifswald.
#
10.1021/jm8013234 CCC: $40.75
2009 American Chemical Society
Published on Web 01/26/2009

Ruthenium Complexes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 917
Figure 2. Synthesis of the dinuclear ruthenium(II)-arene complexes 1c-6c. For items marked with an asterisk (*) see ref 37 for synthetic procedure.
recently organometallic ruthenium(II)-arene complexes were
reported to exhibit antitumoral activity.26-28 RAPTA and
ethylenediamine compounds (see Figure 1) are the most widely
studied representatives of this compound class. RAPTA com-
plexes exhibit selective cytotoxicity in TS/A cancer cells relative
to nontumorigenic HBL-100 cells and were found to be, similar
to NAMI-A, active in vivo against lung metastases derived from
a MCa mammary carcinoma in CBA mice and in Ehrlich ascites
carcinoma.27,29 Ethylenediamine (en) complexes with the general
formula [Ru(arene)(en)Cl]+ can be optimized in terms of
biological activity and minimized side effects by variation of
the arene and the nonleaving groups.26 The compounds are
equally potent toward cisplatin-sensitive (A2780) and cisplatin-
resistant human ovarian cancer cells in culture (A2780cis), and
similar results were obtained in vivo.
Figure 3. Time course of UV/vis spectra at λ 325 nm for 4c in dry
methanol and after addition of 10% water (25 °C).
In contrast to multinuclear platinum complexes, analogous
ruthenium compounds have rarely been studied, with just a few
examples to be found in literature.30-36 Herein, we report
structure-activity relationships for an extended series of di-
nuclear pyridinone-derived ruthenium complexes37,38 with dif-
ferent spacer lengths, based on chemical and (bio)analytical
characterization, i.e., octanol/water partition, hydrolytic stability,
reactivity toward biomolecules, and extraordinary in vitro
activity in human tumor cell lines.
that did not change over time (Supporting Information, Figure
1
S1). Time-resolved H NMR spectra of 4c in D2O before and
after the addition of AgNO3, which guaranteed the full hydroly-
sis of the Ru complex, also showed no changes over 5 d
(Supporting Information, Figure S2). These results suggest that
the hydrolysis occurs immediately after dissolution of the
sample. To slow the hydrolysis reaction, 4c was dissolved in
dry methanol and analyzed again by UV/vis (Figure 3, Sup-
porting Information, Figure S3). After a few measurement
cycles, 10% of water was added and the reaction was followed
for 10 h, showing a significant change of the absorption bands
over time after the addition of water.
With a chloride selective electrode the release of the chlorido
ligands from 4c in water was followed over time. Immediately
after preparation of a 0.5 mM solution, the chloride ion
concentration was observed to be 0.53 mM and increased rapidly
until the complete release of the second chloride ion (Figure
4a). In order to slow this process, 4c was dissolved in 0.5 mL
of methanol and added to water (Figure 4b). For 0.5 and 1 mM
methanolic solutions, the free chloride concentration was
measured immediately after addition to water, and within a few
minutes chloride concentrations of 1 and 2 mM, respectively,
were detected, indicating full aquation of the complex. These
findings are in good agreement with those obtained by 1H NMR
and UV/vis. In contrast to the analogous mononuclear complex
chlorido(maltolato-κO2)(η6-p-cymene)ruthenium(II) 8, there was
no indication of the formation of dimeric hydroxido-bridged
species.39
Results and Discussion
Synthesis and Characterization of Dinuclear Ru(II)-Arene
Complexes. To complete a series of recently developed di-
nuclear Ru(II)-arene compounds37 with the purpose of estab-
lishing more detailed structure-activity relationships, the Ru
complexes 1c, 3c, and 5c with varying aliphatic spacers were
prepared in a similar way as reported earlier.37 The complexes
were obtained from the reaction between [(η6-p-cymene)RuCl2]2
and sodium methoxide-deprotonated bis(3-hydroxy-2-methyl-
4-pyridinon-1-yl)alkanes 1b, 3b, and 5b (alkane ) ethane 1b,
butane 2b, octane 2c) in good yields (41%, 58%, and 66%,
respectively, Figure 2). The complexes were characterized by
1H and 13C NMR spectroscopy, MS, and elemental analysis.
As reported earlier for 2c, 4c, and 6c, only one set of signals
1
was observed in the H NMR spectra. Electrospray ionization
mass spectra contained as the most abundant peaks doubly
charged [M - 2Cl]2+ ions, while lower intensity peaks were
assignable to [M - Cl]+ and [M + Na]+.
Hydrolytic Stability. To investigate the hydrolytic stability
of this type of complex, the behavior of 4c in water was followed
spectroscopically by UV/vis and NMR methods, and the chloride
release was determined with a chloride selective electrode. A
kinetic UV/vis experiment of 4c in water was performed over
2 d. Two absorption bands at λ 314 and 224 nm were observed
pKa Determination. The pKa values of 2c, 4c, and 6c were
determined by NMR titration and are exemplary for the whole
series of compounds. The titration curves were plotted as a
function of the chemical shift of the aromatic protons of the
p-cymene arene group versus the measured pD values (Sup-

918 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Mendoza-Ferri et al.
Figure 4. Chloride ion concentration over time of (a) 0.5 mM 4c solution in water, (b) 0.5 and 1 mM 4c solutions prepared in 0.5 mL of methanol
and added to 4.4 mL of water (containing final 5 M NaNO3) at 25 °C.
Table 1. pKa Values Obtained by the pD Titration
Table 2. The log Poct/water Values, Determined by Using the Shake Flask
Method and Analysis by ICP-MS and UV/Visible Spectroscopy, and
Cytotoxicity of Complexes 1c-6c Compared to the Mononuclear
Complexes 7 and [Ru(cym)(mal)Cl] 8 in SW480 and A2780 Human
Cancer Cells, Determined by the MTT Assay
complex
pKa values
2c
4c
6c
9.70 ( 0.03
9.60 ( 0.02
9.83 ( 0.06
9.23
log Poct/water
IC50, µM
[RuCl(cym)(mal)] 8a
compd
n
ICP-MS UV/vis
SW480
A2780
a From ref 39.
1c
2
-1.42
-1.39
-1.33
-1.36
-1.28
-1.42 76 ( 4
84 ( 3
2ca
3c
3
4
6
8
-1.40 62 ( 14a
-1.38 28 ( 1
-1.34 26 ( 8a
-1.23 2.5 ( 0.2
25 ( 2a
41 ( 2
porting Information, Figure S4). The pKa values were obtained
from the inflection points of the curves and corrected to consider
the difference between H2O and D2O. The pKa values for 2c,
4c, and 6c indicate that the complexes are present as aquated
species under biological conditions (pH 7.4), with the linker
length having only a very small influence on the pKa (see
Table 1). The pKa values obtained range from 9.60 to 9.83 and
are close to the one of the mononuclear analogue chlorido(ma-
ltolato-κO2)(η6-p-cymene)ruthenium(II), i.e., 9.23.39 The coor-
dination of strong electron donating ligands such as maltol-
derived pyridinone to a ruthenium(II) center facilitates the
formation of aqua over hydroxido complexes. Note that the pKa
values can be easily modified by replacing the O,O chelating
ligands by ethylenediamine, which leads to a decrease of the
pKa values by about 1 unit (pKa ≈ 8).39 Replacing the ruthenium
center by osmium results in a pKa being 1.6 units lower than
that of the Ru(II) species.39
Determination of Lipophilicity: log Poct/water Values. The
lipophilicity of the complexes 1c-6c was measured as the n-
octanol/water partition coefficient, log P, by applying the shake
flask method40 and quantification of the ruthenium contents in
the aqueous and octanol phases with ICP-MS and UV/vis,
respectively. Because of the fast hydrolysis of the compounds
in aqueous solution, the log Poct/water values that were determined
were actually those of the aqua complexes, which were in the
range from -1.42 to -0.56 (see Table 2). These values show
a marked correlation to the spacer length linking the pyridinone
moieties.
4ca
5c
30 ( 6a
5.7 ( 0.5
6ca
7
12 -0.56
-0.58 0.29 ( 0.05a 1.5 ( 0.3a
42 ( 1
>100
88 ( 12
[RuCl(cym)(mal)] 8
>100b
a From ref 37. b From ref 39.
former compound is roughly equieffective in the ovarian and
colon cancer cell lines, while the latter reveals large differences
in their sensitivity. In other words, the cytotoxic effects of 6c
are to a greater extent dependent on the cell type whereas 1c
exerts rather unspecific effects. This same trend is apparent in
seven of the eight cell lines shown in Table 3; i.e., 6c is more
active than 4c and 2c. As with the smaller panel of cells, the
eight cell line panel shows a large difference in sensitivity to
6c, with a more than 10-fold difference in IC50 values between
MCF-7 and KYSE70 cells.
Chain length-activity relationships correlate to some extent
with the hydrophobicity of the complexes as reflected by their
log P values (Table 2), although no quantitative relationship
could be established. As highlighted above, the complexes attain
a more hydrophobic character with increasing chain length
between the two pyridinone moieties. Hydrophobicity may
contribute to an increased uptake of the complex into the cells,
thereby enhancing antiproliferative activity. The fact that no
direct correlation to lipophilicity was observed supports the idea
that other effects are also important for the mode of action.
Comparison of the in vitro activity with that of cisplatin,
oxoplatin [a Pt(IV) cisplatin precursor with two hydroxido
ligands in the axial positions] and oxaliplatin shows that the
most cytotoxic compound 6c is as active as oxaliplatin in the
colon carcinoma cell line SW480 and more active than cisplatin
and the anticancer drug candidate KP1019 in A2780 cells.37 In
five of the eight cell lines LCLC-103, A-427, RT-4, MCF-7,
DAN-G, 5637, SISO, and KYSE70, 6c is less active than
cisplatin but more active than oxoplatin (Table 3).
Cytotoxicity in Cancer Cell Lines. The antiproliferative
activity of the dinuclear complexes was determined in the two
human cancer cell lines SW480 (colon) and A2780 (ovary) by
means of the colorimetric MTT assay and in the other cell lines
with the crystal violet assay. The IC50 values are listed in Tables
2 and 3, and representative concentration-effect curves in
A2780 cells are shown in Figure 5. Chain length-activity
relationships in the human cell lines are similar, indicating that
cytotoxicity increases with the chain length of the alkane spacer
from IC50 values in the high 10-5 M range in the case of 1c,
decreasing to very low micromolar or even submicromolar
concentrations in the case of 6c (Table 2). Remarkably, the
Comparison of the dinuclear complexes with [RuCl(cym)-
(mal)] 8 and chlorido[2-methyl-3-(oxo-κO)-1-propyl-4(1H)-
pyridinonato-κO4](η6-p-cymene)ruthenium(II) 7 (see supporting

Ruthenium Complexes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 919
Figure 5. Concentration-effect curves of the dinuclear Ru(II)-ar-
ene complexes 1c-6c in A2780 cells, as obtained by the MTT assay
(96 h exposure).
information for structures of 7 and 8), a mononuclear analogue
of 4c, reveals that a change from the maltolato to a pyridinonato
ligand clearly increases the antiproliferative activity. Most
notably, the linking of two metal centers results in a more than
2-fold enhancement of activity in both cell lines (Table 2).
In three oxoplatin-resistant cell lines, 5637-oxo, SISO-oxo,
and KYSE70-oxo, which are also completely cross-resistant to
cisplatin, no cross-resistance to 6c was observed (Table 3). In
fact, the cell lines 5637-oxo and KYSE70-oxo are more than
10-fold more sensitive to 6c than the parent cell lines 5637 and
KYSE70, respectively. A dramatic reversal of resistance was
not apparent for 2c and 4c, however. These data provide
evidence that 6c acts by a mechanism that is amplified when
cells become resistant to oxoplatin/cisplatin.
Reactivity toward Proteins and Nucleotides
Protein Affinity. Proteins are known to be primary targets
for anticancer metallodrugs.20 With the goal of gaining deeper
insight into the mode of action of the complexes, mass
spectrometric experiments were conducted to elucidate the
affinity toward transferrin (Tf), ubiquitin (Ub), and cytochrome
c (Cyt-c). The stoichiometry of the binding to transferrin, which
is the most important iron transport protein in the body41 and
may thus act as a selective vehicle for ruthenium and other
antitumor metal compounds to the iron-demanding tumor cells,
as well as to Ub42 and Cyt-c,43 which were chosen as model
cellular proteins, was evaluated. Complex 2c was investigated
as a representative complex because it has good aqueous
solubility while still having a considerable spacer length between
the two Ru moieties. Samples containing varying drug-to-protein
ratios from 1:1 to 8:1 were prepared and analyzed by electro-
spray ionization mass spectrometry (ESI-MS). In the cases of
Ub and Cyt-c, no adduct formation could be observed even at
an 8-fold excess of the drug (data not shown). This observation
also argues against unspecific gas phase reactions that could
feign adduct formation.
By contrast, adduct formation of 2c with Tf was already
observed at a 1:1 ratio. A molecular weight of 79 565 ( 16 Da
was determined for the protein, which is in good accordance
with the literature.44 A mass increase of approximately 760 Da
was assigned to a conjugate formed with the dechlorinated
complex, whereas a mass increase of approximately 1520 Da
was assigned to the addition of a second dechlorinated species
(Figure 6). Interestingly, no adducts corresponding to the binding
of more than two complex molecules (mass increase of
approximately 1520 Da, exchange of chlorido ligands) were
detected, even at 8-fold excess of the complex. One could
speculate that specific binding of the Ru moieties to the iron-
binding pockets of the serum protein results in very stable

920 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Mendoza-Ferri et al.
Figure 6. Recorded (left) and maximum entropy deconvoluted (right) mass spectra of transferrin with 2-fold excess of 2c, recorded after 30 min
of incubation in 20 mM ammonium carbonate buffer at 37 °C and pH 7.4. Peak 1 corresponds to the protein itself, peak 2 to the monoadduct (mass
increase of ∼760 Da), peak 3 to the bisadduct (mass increase of ∼1520 Da).
conjugates, which might also be enhanced by electrostatic
attraction: the calculated pI of the protein is 6.81,45 resulting
in an overall negative charge under physiological conditions
(pH 7.4), whereas the complex is doubly positively charged
because of hydrolysis. An exact elucidation of the structure was
not possible because of the inaccuracy of the mass spectrometer
equipped with an ion trap analyzer; thus, a higher resolution
instrument is required.
Affinity toward DNA and DNA Model Compounds. DNA
is regarded as one of the most important targets for Pt anticancer
compounds. Binding to their preferred target, i.e., guanine
residues, leads to a modification of the DNA secondary structure
and inhibition of transcription and replication.1
The dinuclear complexes 4c and 5c were reacted with the
nucleotides guanosine 5′-monophosphate (GMP), adenosine 5′-
monophosphate (AMP), cytidine 5′-monophosphate (CMP),
Figure 7. 1H NMR spectra for the reaction of 5c with AMP at different
uridine 5′-monophosphate (UMP), and thymidine 5′-mono-
ratios of metal complex/nucleotide in NaClO4/D2O.
phosphate (TMP) at molar ratios of 20:1, 10:1, 5:1, 3.3:1, 2:1,
1.6:1, and 1:1, and the reaction was followed by 1H NMR and
31P NMR spectroscopy. For both complexes similar behavior
was observed: The formation of GMP and AMP adducts
proceeded rapidly and quantitatively up to a ratio of 2:1
(complex/nucleotide), as evidenced by a shift of the H8 proton
of GMP from approximately 8.1 to 7.8 ppm and of the H8 and
H2 protons of AMP from 8.4 to 8.7 and from 8.2 to 8.3,
respectively.46 Coordination of AMP resulted in a doublet
formation of the signals assigned to the purine protons, and for
both AMP and GMP the arene and pyridone regions (6-7 and
5-6 ppm, respectively) of the 1H NMR spectra contained a high
number of signals that change over time (Figure 7 for the
Figure 8. Deconvoluted ESI mass spectrum for a mixture of 4c and
a DNA 13-mer (molar ratio 1:1; rB ) 0.15) after 10 min of incubation
time.
reaction of AMP with 5c). For CMP, TMP, and UMP no
reaction was observed, as also reported for other metal-arene
compounds.39 In the 31P NMR spectra no indication for
coordination of the phosphate moiety to the metal center was
observed.
Furthermore, the affinity of 4c to a DNA 13-mer (3921 Da)
was studied by means of ESI-MS, and the reaction was followed
over 24 h (for experimental details, see Materials and Methods).
Different DNA/complex ratios were prepared, and the ruthen-
ation increased with the rB. For example, at rB ) 0.15 the 13-
mer and an adduct signal, assignable to 13-mer + [4c - 2Cl]
(4721 Da), were observed at a relative intensity of approximately
1:1 (Figure 8). When rB > 0.1, the formation of a bis-adduct
was attained (13-mer + [2 4c - 4Cl], 5521 Da). At rB ) 0.77,
the ratio between these two adducts was found to be 1:1, with
no unruthenated 13-mer being detectable. It was not possible
to further increase the concentration of 4c, since this induced
the precipitation of the 13-mer. Furthermore, precipitation was
observed in all samples with rB > 0.1 after incubation for more
than 24 h, and in the remaining mother liquor neither adduct
nor unreacted 13-mer was detectable. As observed for the
nucleotides AMP and GMP, the reaction proceeded very rapidly
and no changes over time were observed. It was also considered
that the dinuclear complex could cross-link two 13-mer mol-
ecules; however, such species were not observed in the mass
spectra but cannot be excluded to be present in the precipitate.
The affinity of dinuclear Ru(II) complexes toward DNA was
assayed utilizing plasmid DNA, which showed that the addition
of 2c and 4c induced slight unwinding of supercoiled plasmid
DNA. The reaction of 2c and 4c with calf thymus DNA showed
a high degree of ruthenation (nearly 50% of the applied amount).

Ruthenium Complexes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 921
Synthesis. General Procedure for 1a, 3a, and 5a. Sodium
hydroxide (1.40 g, 35.0 mmol) was added to a solution of
3-benzyloxy-2-methyl-4-pyrone (10.0 g, 46.2 mmol) and 1,n-
diamine (15.4 mmol) in a methanol-water mixture (2:1, 210 mL).
The reaction mixture was refluxed for 48 h and then allowed to
cool to room temperature. The product was extracted with dichlo-
romethane (3 × 50 mL), and afterward the solvent was removed
in vacuum. The pure product was isolated by column chromato-
graphy on silica gel.
Similar observations were made in a more detailed study on
the DNA binding, most notably with a high degree of
DNA-protein and DNA interduplex cross-linking, which is
again dependent on the spacer length.47
The linearization of plasmid DNA by 2c and 6c was assayed
by gel electrophoresis. For this purpose, the two restriction
enzymes BamH1 and HindIII, which are known to cleave DNA
sequence specifically at GG and AA sites, respectively, were
added to 48 h preincubated complex-pUC18 plasmid mixtures.
In contrast to traditional platinum anticancer agents,48 both 2c
and 6c do not inhibit the linearization of the plasmid DNA.
1,2-Bis[3-benzyloxy-2-methyl-4(1H)-pyridinon-1-yl]ethane
(1a). From NaOH (1.40 g, 35.0 mmol), 3-benzyloxy-2-methyl-4-
pyrone (10.0 g, 46.2 mmol), and 1,2-ethylenediamine (0.92 g, 15.4
mmol) 1a was obtained. Yield 2.03 g (29%), mp 198-199 °C. Anal.
1
(C28H28N2O4 ·2/3H2O) C, H, N. MS (ESI+) m/z 457 [M + H]+. H
Conclusions
NMR in MeOH-d4: δ ) 2.08 [s, 6H, CH3], 4.29 [s, 4H, CH2-N],
5.12 [s, 4H, CH2-Ph], 6.36 [d, 2H, CH-CdO, 3J ) 7.3 Hz], 7.17
[d, 2H, CH, 3J ) 7.6 Hz], 7.35-7.42 [m, 10H, CHarom]. 13 C NMR
in MeOH-d4: δ ) 11.7 [CH3], 53.2 [CH2-N], 73.4 [CH2-Ph],
116.8 [CH-CdO], 128.5 [CH], 128.5 [CH], 129.2 [CH], 137.4
[Carom], 140.0 [CH], 143.5 [C-CH3], 146.2 [C-O], 174.2 [CdO].
1,4-Bis[3-benzyloxy-2-methyl-4(1H)-pyridinon-1-yl]butane
(3a). From NaOH (1.40 g, 35.0 mmol), 3-benzyloxy-2-methyl-4-
pyrone (10.0 g, 46.2 mmol), and 1,4-diaminobutane (1.36 g, 15.4
mmol) 3a was obtained. Yield: 3.91 g (52%), mp 202-204 °C.
Anal. (C30H32N2O4 ·1/3H2O) C, H, N. MS (ESI+) m/z 485 [M +
H]+. 1H NMR in MeOH-d4: δ ) 1.65 [brs, 4H, CH2], 2.17 [s, 6H,
CH3], 3.96 [brs, 4H, CH2-N], 5.11 [s, 4H, CH2-Ph], 6.48 [d, 2H,
Ruthenium anticancer drug candidates tend to be significantly
less toxic both in vitro and in vivo than the established platinum
complexes. Therefore, there exists the potential to increase
cytotoxicity without damaging healthy tissue. One proven option
for increasing the cytotoxicity of platinum compounds is to link
several platinum atoms by diaminoalkane ligands, resulting in
compounds such as BBR3464 that are even active in cisplatin-
resistant cell lines.
Dinuclear Ru(II)-arene compounds with the Ru centers
acting cooperatively in human tumor cell lines have been
developed and (bio)analytically characterized with respect to
structure-activity relationships. As usual for many organome-
tallic ruthenium compounds, rapid hydrolysis was observed, with
the aqua complexes exhibiting pKa values of about 9.7. Similar
to other Ru complexes, affinity for transferrin was observed,
but surprisingly no interaction with the smaller cellular proteins
ubiquitin and cytochrome c was detected. The compounds were
found to react rapidly with DNA and model nucleotides, as
3
CH-CdO, J ) 7.0 Hz], 7.32-7.34 [m, 10H, CHarom], 7.67 [d,
3
2H, CH, J ) 7.5 Hz]. 13C NMR in MeOH-d4: δ ) 11.8 [CH3],
27.2 [CH2CH2-N], 53.5 [CH2-N], 73.4 [CH2-Ph], 116.4
[CH-CdO], 128.4 [CH], 128.4 [CH], 129.3 [CH], 137.4 [Carom],
140.0 [CH], 143.9 [C-CH3], 146.0 [C-O], 173.8 [CdO].
1,8-Bis[3-benzyloxy-2-methyl-4(1H)-pyridinon-1-yl]oc-
tane (5a). From NaOH (1.40 g, 35.0 mmol), 3-benzyloxy-2-methyl-
4-pyrone (10.0 g, 46.2 mmol), and 1,8-diaminooctane (2.22 g, 15.4
mmol) 5a was obtained. Yield: 4.04 g (49%), mp 96-98 °C. Anal.
1
observed by H NMR spectroscopy, DNA precipitation, mass
spectrometry, and gel electrophoresis. Their in vitro anticancer
activity in several human tumor cell lines correlates with spacer
length and lipophilicity. Interestingly, two cell lines resistant
to a cisplatin analogue, oxoplatin, are more sensitive to the
dinuclear Ru(II)-arene 6c than the native cell lines.
1
(C34H40N2O4 ·2H2O) C, H, N. MS (ESI+) m/z 541 [M + H]+. H
NMR in MeOH-d4: δ ) 1.33 [brs, 8H, CH2], 1.64-1.67 [m, 4H,
3
CH2CH2-N], 2.17 [s, 6H, CH3], 3.94-3.98 [t, 4H, CH2-N, J )
3
7.5 Hz], 5.10 [s, 4H, CH2-Ph], 6.48 [d, 2H, CH-CdO, J ) 7.6
Hz], 7.33-7.41 [m, 10H, CHarom], 7.69 [d, 2H, CH, 3J ) 7.5 Hz].
13C NMR in MeOH-d4: δ ) 11.8 [CH3], 26.1 [CH2(CH2)3-N], 29.1
[CH2(CH2)2-N], 30.5 [CH2CH2-N], 54.3 [CH2-N], 73.4
[CH2-Ph], 116.3 [CH-CdO], 128.3 [CH], 128.4 [CH], 129.4
[CH], 137.4 [Carom], 140.2 [CH], 144.0 [C-CH3], 145.9 [C-O],
173.7 [CdO].
Materials and Methods
All reactions were carried out in dry solvents and under argon
atmosphere. Bis[dichlorido(η6-p-cymene)ruthenium(II)]49 and
3-benzyloxy-2-methyl-4-pyrone50 as well as 2c, 4c, 6c,37 chlo-
rido[2-methyl-3-(oxo-κO)-1-propyl-4(1H)-pyridinonato-κO4](η6-
p-cymene)ruthenium(II) 7,51 and chlorido(maltolato-κO2)(η6-p-
cymene)ruthenium(II) 839 were prepared according to literature
procedures. Purchased chemicals were used without further puri-
fications. Melting points were determined with a Bu¨chi B-540
apparatus and are uncorrected. Elemental analyses were carried out
with a Perkin-Elmer 2400 CHN elemental analyzer at the Mi-
croanalytical Laboratory of the University of Vienna. NMR spectra
were recorded at 25 °C on a Bruker Avance DPX400 spectrometer
(Ultrashield magnet) at 400.13 MHz (1H) and 100.63 MHz (13C)
in MeOH-d4, DMSO-d6, or D2O/CF3COOH (9: 1). Electrospray
ionization mass spectrometry (ESI-MS) was performed on a Bruker
Esquire3000 instrument (Bruker Daltonics, Bremen, Germany), and
theoretical and experimental isotope distributions were compared.
Inductively coupled plasma mass spectrometric (ICP-MS) analyses
were made on an Agilent 7500ce quadrupole mass spectrometer
(Agilent, Waldbronn, Germany) and ultraviolet-visible spectro-
scopic (UV/vis) measurements on a Perkin-Elmer Lambda 650
instrument (from 500 to 200 nm). Apotransferrin (97%, lot
074K1370), ubiquitin (from bovine red blood cells, min 90%, lot
075K7405), cytochrome c (from horse heart, 99%, lot 065K7001),
and ammonium bicarbonate (purum, pa) were products of Sigma-
Aldrich (Vienna, Austria). High-purity water used for the MS
experiments was obtained from a Millipore Synergy 185 UV
Ultrapure Water system (Molsheim, France).
General Procedure for 1b, 3b, and 5b. Hydrogen was passed
through a mixture of 1,n-bis[3-benzyloxy-2-methyl-4(1H)-pyridi-
non-1-yl]alkane and palladium on activated carbon (10% Pd) in
100% acetic acid. The conversion was monitored by means of TLC,
and the reaction was terminated when the spot of the starting
compound disappeared. The catalyst was filtered off, the solvent
was removed, and the product was dried in vacuum.
1,2-Bis[3-hydroxy-2-methyl-4(1H)-pyridinon-1-yl]ethane (1b).
From 1a (0.50 g, 1.10 mmol) and palladium on activated carbon
(75 mg, 10% Pd) in acetic acid (100%, 40 mL) 1b was obtained.
Yield: 0.26 g (86%), mp 300-302 °C (dec). Anal. (C14H16N2O4 ·
7/6H2O) C, H, N. MS (ESI+) m/z 277 [M + H]+. 1H NMR in D2O/
CF3COOH: δ ) 0.79 [s, 6H, CH3], 3.06 [s, 4H, CH2], 5.37 [d, 2H,
CH-CdO, 3J ) 7.0 Hz], 5.89 [d, 2H, CH, 3J ) 7.1 Hz]. 13C NMR
in D2O/CF3COOH: δ ) 10.1 [CH3], 53.2 [CH2-N], 110.1
[CH-CdO], 136.3 [CH], 140.1 [C-CH3], 142.7 [C-O], 158.0
[CdO].
1,4-Bis[3-hydroxy-2-methyl-4(1H)-pyridinon-1-yl]butane (3b).
From 3a (0.80 g, 1.65 mmol) and palladium on activated carbon
(80 mg, 10% Pd) in acetic acid (100%, 40 mL) 3b was obtained.
Yield: 0.20 g (40%), mp 296-298 °C (dec). Anal. (C16H20N2O4)
C, H, N. MS (ESI+) m/z 305 [M + H]+. 1H NMR in D2O/
CF3COOH: δ ) 0.70 [brs, 4H, CH2], 1.34 [s, 6H, CH3], 3.10 [brs,
3
4H, CH2-N], 5.92 [d, 2H, CH-CdO, J ) 7.0 Hz], 6.64 [d, 2H,
3
CH, J ) 7.1 Hz]. 13C NMR in D2O/CF3COOH: δ ) 10.5 [CH3],

922 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Mendoza-Ferri et al.
25.5 [CH2CH2-N], 54.8 [CH2-N], 110.3 [CH-CdO], 136.6 [CH],
140.8 [C-CH3], 142.6 [C-O], 157.5 [CdO].
Determination of log P Values. The shake flask method was
used to determine the n-octanol/water partition coefficient of the
dinuclear ruthenium(II)-arene compounds.40 UV/vis spectroscopy
and ICP-MS were used to determine the concentration of the
ruthenium complexes in the organic and aqueous phase, respec-
tively. For UV/vis quantification, the wavelength of the absorption
maximum was determined for each complex (λ 327, 325, and 325
nm for 1c, 3c, and 5c, respectively), and an external calibration
was performed in the range from 1 to 100 µM with standards
prepared in n-octanol. The ruthenium concentration in the aqueous
phase was determined by ICP-MS using external calibration from
1 to 30 ppb. Both 101Ru and 102Ru isotopes were employed for
quantification and gave essentially the same results. All solutions
were prepared in 5% HNO3 (HNO3, pa from Fluka, further purified
with a quartz sub-boiling system from Milestone-MLS GmbH,
Leutkirch, Germany; 18.2 MΩ H2O was obtained from a Synergy
185, Millipore, Bedford, MA), and 1 ppb of 185Re was added for
internal standardization to the samples and standard solutions.
Determination of pKa Values. The pH values of the NMR
samples of the aqua complexes (prepared in D2O and stirred
overnight in the presence of 2 equiv of AgNO3) were measured
with an Eco Scan pH meter equipped with a combination pH glass
electrode-microelectrode (Orion 9826BN) and calibrated with
standard buffer solutions of pH 4, 7, and 10. The pH titration was
performed by addition of sodium deuteroxide (40% in D2O) or
D-nitric acid (65% in D2O), and the curves were fitted to the
Henderson-Hasselbalch equation by KALEIDAGRAPH (version
4.03) software, assuming that the observed chemical shifts are
weighted averages according to the populations of the protonated
and deprotonated species. The pKa values determined experimentally
were corrected by using eq 1,52 which converts the pKa value
determined in D2O (pKa*) into the corresponding pKa in aqueous
solutions.
1,8-Bis[3-hydroxy-2-methyl-4(1H)-pyridinon-1-yl]octane (5b).
From 5a (1.0 g, 1.85 mmol) and palladium on activated carbon
(100 mg, 10% Pd) in acetic acid (100%, 20 mL) 5b was obtained.
Yield: 0.45 g (68%), mp 261-263 °C (dec). Anal. (C20H28N2O4 ·
2/3H2O) C, H, N. MS (ESI+) m/z 361 [M + H]+. 1H NMR in D2O/
CF3COOH: δ ) 1.07 [brs, 8H, CH2], 1.57 [m, 4H, CH2CH2-N],
3
2.35 [s, 6H, CH3], 4.08 [t, 4H, CH2-N, J ) 7.5 Hz], 6.89 [d, 2H,
CH-CdO, 3J ) 6.5 Hz], 7.80 [d, 2H, CH, 3J ) 7.0 Hz]. 13C NMR
in D2O/CF3COOH: δ ) 12.3 [CH3], 25.4 [CH2(CH2)3-N], 28.2
[CH2(CH2)2-N], 29.6 [CH2CH2-N], 57.1 [CH2-N], 111.0
[CH-CdO], 138.6 [CH], 142.8 [C-CH3], 142.8 [C-O] 158.1
[CdO].
General Procedure for 1c, 3c, and 5c. A solution of bis[dichlo-
rido(η6-p-isopropyltoluene)ruthenium(II)] in methanol was added
to a suspension of 1,n-bis[3-hydroxy-2-methyl-4(1H)-pyridinon-1-
yl]alkane and sodium methoxide in methanol. The reaction mixture
was stirred at room temperature under argon atmosphere for 2 days.
The unconverted ligand was removed by filtration, and the solvent
was evaporated under vacuum. The pure complex was obtained by
extraction with a dichloromethane-diethyl ether (2:1) mixture. The
solvents were removed, and the orange-red compound was dried
in vacuum.
1,2-Bis{chlorido[3-(oxo-KO)-2-methyl-4(1H)-pyridinonato-
KO4](η6-p-cymene)ruthenium(II)}ethane (1c). From bis[dichlo-
rido(η6-p-cymene)ruthenium(II)] (155 mg, 0.25 mmol), 1b (100 mg,
0.36 mmol), and sodium methoxide (43 mg, 0.80 mmol), 1c was
obtained. Yield: 85 mg (41%), mp 160-164 °C (dec). Anal.
(C34H42N2O4Ru2Cl2) C, H, N. MS (ESI+) m/z 781 [M-Cl]+ and
373 [M - 2Cl]2+
.
1H NMR in MeOH-d4: δ ) 1.33 [d, 12H,
3
(CH3)2CH, J ) 6.8 Hz], 2.28 [s, 6H, CH3], 2.31 [s, 6H, CH3],
2.80-2.88 [m, 2H, CH(CH3)2], 4.44 [s, 4H, CH2-N], 5.43 [d, 4H,
3
3
CHarom, J ) 5.8 Hz], 5.65 [d, 4H, CHarom, J ) 5.8 Hz], 6.31 [d,
3
3
pKa ) 0.929pKa* + 0.42
(1)
2H, CH-CdO, J ) 6.8 Hz], 6.87 [d, 2H, CH, J ) 6.8 Hz]. 13C
NMR in MeOH-d4: δ ) 10.6 [CH3], 17.5 [CH3], 21.6 [(CH3)2CH],
31.4 [CH(CH3)2], 53.9 [CH2-N], 77.9 [CH], 79.7 [CH], 96.2
[Carom], 97.1 [Carom], 109.7 [CH-CdO], 133.0 [CH], 133.9
[C-CH3], 159.9 [C-O], 175.1 [CdO].
Determination of the Chloride Ion Concentration. The
chloride ion concentration after dissolution of 4c in water or in
water/methanol was recorded with a CyberScan 2100 pH/ion meter
over time at 25 °C. The instrument was equipped with an Eutech
chloride ion combination epoxy-body electrode calibrated from 10
to 1000 ppm. The ionic strength of the samples was maintained
constant by addition of 0.1 mL of 5 M NaNO3 solution to 4.9 mL
of sample solution (0.5 or 1 mM).
1,4-Bis{chlorido[3-(oxo-KO)-2-methyl-4(1H)-pyridinonato-
KO4](η6-p-cymene)ruthenium(II)}butane (3c). From bis[dichlo-
rido(η6-p-cymene)ruthenium(II)] (109 mg, 0.18 mmol), 3b (60 mg,
0.20 mmol), and sodium methoxide (23 mg, 0.43 mmol), 3c was
obtained. Yield: 85 mg (57%), mp 280-282 °C (dec). Anal.
(C36H46N2O4Ru2Cl2) C, H, N. MS (ESI+) m/z 387 [M - 2Cl]2+
.
DNA and Nucleotide Binding Studies
1H NMR in MeOH-d4: δ ) 1.33 [d, 12H, (CH3)2CH, 3J ) 6.5 Hz],
1.72 [brs, 4H, CH2CH2-N], 2.27 [s, 6H, CH3], 2.45 [s, 6H, CH3],
2.82-2.89 [m, 2H, CH(CH3)2], 4.07 [brs, 4H, CH2-N], 5.43 [d,
4H, CHarom, 3J ) 6.0 Hz], 5.66 [d, 4H, CHarom, 3J ) 6.0 Hz], 6.48
Gel Electrophoresis. The plasmid pUC18 (2686 bp) was
transformed in XL1 blue E. coli bacteria, isolated, purified
according to standard procedures,53 and dissolved in TE buffer
(10 mM Tris-Cl, pH 7.4, 1 mM Na2EDTA).
3
3
[d, 2H, CH-CdO, J ) 6.5 Hz], 7.38 [d, 2H, CH, J ) 7.0 Hz].
13C NMR in MeOH-d4: δ ) 10.8 [CH3], 17.5 [CH3], 21.6
[(CH3)2CH], 27.4 [CH2CH2-N], 31.4 [CH(CH3)2], 54.2 [CH2-N],
77.9 [CH], 79.6 [CH], 96.0 [Carom], 98.9 [Carom], 109.4 [CH-CdO],
133.6 [CH], 134.2 [C-CH3], 156.4 [C-O], 174.2 [CdO].
In order to investigate the type of interaction between plasmid
DNA and the dinuclear complexes, an amount of 100 ng of
pUC18 was incubated with 2c and 6c, applying a concentration
gradient from 0 to 50 µM in 20 mM phosphate buffer at pH
7.4 and 37 °C. After 48 h of incubation in the dark, the split
sample was incubated with the restriction enzymes BamH1 and
HindIII for 2 h at 37 °C. Afterward, both the digested and the
nondigested samples were loaded on a 1% agarose gel and
separated at 25 °C and 100 V in TAE buffer (0.04 M Tris-
acetate, 1 mM EDTA, pH 7.0). The gel was stained with
ethidium bromide (0.2 µg/mL) in TAE buffer for 30 min and
visualized by UV.
Mass Spectrometry. For the MS studies, the non-self-
complementary single-stranded DNA 13-mer 5′-ATC TGT TTG
TCT T-3′ (3921 Da; Midland Certified Reagents, Midland TX)
was incubated with 4c in H2O at DNA (40 µM), with complex
ratios ranging from 5:1 (rB ) 0.031; rb is the ruthenium:
nucleotide ratio) to 1:5 (rB ) 0.77). Before use, the purified
13-mer was further desalted by using a custom built dialysis
1,8-Bis{chlorido[3-(oxo-KO)-2-methyl-4(1H)-pyridinonato-
KO4](η6-p-cymene)ruthenium}octane (5c). From bis[dichlorido(η6-
p-cymene)ruthenium(II)] (76 mg, 0.12 mmol), 5b (50 mg, 0.14
mmol), and sodium methoxide (17 mg, 0.31 mmol), 5c was
obtained. Yield: 73 mg (65%), mp 288-290 °C (dec). Anal.
(C40H54N2O4Ru2Cl2) C, H, N. MS (ESI+) m/z 865 [M-Cl]+ and
415 [M - 2Cl]2+. 1H NMR in MeOH-d4: δ ) 1.33-1.35 [m, 20H,
CH2(CH2)3-N, CH2(CH2)2-N,(CH3)2CH], 1.71 [m, 4H,
CH2CH2-N], 2.28 [s, 6H, CH3], 2.49 [s, 6H, CH3], 2.82-2.89 [m,
3
2H, CH(CH3)2], 4.06 [t, 4H, CH2-N, J ) 7.5 Hz], 5.45 [d, 4H,
3
3
CHarom, J ) 6.0 Hz], 5.67 [d, 4H, CHarom, J ) 6.0 Hz], 6.51 [d,
2H, CH-CdO, J ) 6.5 Hz], 7.42 [d, 2H, CH, J ) 7.0 Hz]. 13C
NMR in MeOH-d4: δ ) 10.8 [CH3], 17.5 [CH3], 21.6 [(CH3)2CH],
26.1 [CH2(CH2)3-N], 29.0 [CH2(CH2)2-N], 30.6 [CH2CH2-N],
31.4 [CH(CH3)2], 54.9 [CH2-N], 78.0 [CH], 79.6 [CH], 96.0
[Carom], 99.0 [Carom], 109.3 [CH-CdO], 133.6 [CH], 134.1
[C-CH3], 159.6 [C-O], 174.0 [CdO].
3
3

Ruthenium Complexes
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4 923
chamber equipped with a hollow microdialysis fiber with a 13
kDa molecular weight cut-off (MWCO) from Spectrum Labo-
ratories (Rancho Dominguez, CA) in 25 mM ammonium acetate
solution. The desalted oligonucleotide was lyophilized to dryness
and reconstituted in diionized water (18 MΩ).
obtained from the German Collection of Microorganisms and
Cell Culture (DSMZ, Braunschweig, FRG). The three oxoplatin-
resistant cell lines were established in our laboratories (Greif-
swald) through weekly exposure of cells to increasing concen-
trations of oxoplatin over a period of several months. Cells were
grown in culture flasks (Sarstedt, Germany) as adherent mono-
layer cultures in 90% RPMI medium supplemented with 10%
heat-inactivated fetal bovine serum and the antibiotics benzyl-
penicillin and streptomycin. To the medium for the MCF-7 cells,
1 mM sodium pyruvate and 1% nonessential amino acids were
added. Cultures were maintained at 37 °C in a humidified
atmosphere containing 5% CO2.
MTT Assay Conditions. Cytotoxicity was determined by
means of a colorimetric microculture assay (MTT assay, MTT
) 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bro-
mide). For this purpose, SW480 and A2780 cells were harvested
from culture flasks by trypsinization and seeded into 96-well
microculture plates (Iwaki/Asahi Technoglass, Gyouda, Japan)
in densities of 2.5 × 103 and 5.0 × 103 cells/well, respectively,
in order to ensure exponential growth throughout drug exposure.
After a 24 h preincubation, cells were exposed to serial dilutions
of the test compounds in 200 µL/well complete culture medium
for 96 h. At the end of exposure, drug solutions were replaced
by 100 µL/well RPMI 1640 culture medium (supplemented with
10% heat-inactivated fetal bovine serum and 2 mM L-glutamine)
and 20 µL/well MTT solution in phosphate-buffered saline
(5 mg/mL). After incubation for 4 h, the medium/MTT mixtures
were removed and the formazan crystals formed by vital cells
were dissolved in 150 µL of DMSO per well. Optical densities
at λ ) 550 nm were measured with a microplate reader (Tecan
Spectra Classic) by using a reference wavelength of λ ) 690
nm to correct for unspecific absorption. Quantities of vital cells
were expressed as T/C values by comparison to untreated control
microcultures, and 50% inhibitory concentrations (IC50) were
calculated from concentration-effect curves by interpolation.
Evaluation is based on mean values from at least three
independent experiments, each comprising at least six replicates
per concentration level.
Crystal Violet Assay Conditions. This assay has been
described in detail elsewhere.54 Culture conditions were the same
as used in the MTT assay. Briefly, cells were seeded into 96-
well microculture plates (Sarstedt, Germany) in cell densities
of 1.0 × 103 cells/well except for LCLC-103H, which was
seeded at 250 cells/well. After a 24 h preincubation, cells were
treated with test substance for 96 h. Stock solutions of test
substance were prepared to 20 mM in DMF and diluted 1000-
fold in RPMI 1640 culture medium containing 10% fetal calf
serum. Substances that showed a g50% growth inhibition at
20 µM were tested at five serial dilutions in 4 wells/concentra-
tion to determine the IC50 values as described.54 The staining
of the cells was done for 30 min with a 0.02% crystal violet
solution in water followed by a washing out of the excess dye.
Cell bound dye was redissolved in 70% ethanol/water solution,
and the optical densities at λ ) 570 nm were measured with a
microplate reader (Anthos 2010).
The sample mixtures were analyzed immediately after mixing
and after 0.5 and 24 h of incubation with a Waters/Micromass
Qtof-2 (Manchester, U.K.) instrument equipped with a custom
built nanospray source operated in negative ion mode over a
mass range of 500-2000 m/z. Samples were introduced into
the inlet at 1.0 µL/min with a capillary voltage of -1.9 kV and
a cone voltage of 36 V. The source temperature was maintained
constant at 110 °C throughout the experiments. A 1:1 mixture
of methanol/water (with 25 mM ammonium acetate) was used
as the spray solvent. Data were collected and processed by using
the Mass Lynx 4.0 software, and the deconvolution to molecular
mass scale was performed with the maximum entropy (Max
Ent) software supplied with the instrument.
NMR Spectroscopy. For studying the reaction of the
dinuclear complex 5c (1 mM) with nucleotides, the metal
complex was reacted with guanosine 5′-monophosphate (GMP),
adenosine 5′-monophosphate (AMP), cytidine 5′-monophosphate
(CMP), uridine 5′-monophosphate (UMP), and thymidine 5′-
monophosphate (TMP) at molar ratios of 20:1, 10:1, 5:1, 3.3:
1, 2:1, 1.6:1, and 1:1 in 10 mM NaClO4 in D2O, and the reaction
1
was followed by H NMR and 31P NMR spectroscopy up to 7
days.
Protein Binding Studies. An Esquire3000 ion trap mass
spectrometer (Bruker Daltonics, Bremen, Germany), equipped
with an orthogonal ESI ion source, was used for mass
spectrometric determination of the complex-protein interac-
tions. The instrument was operated in positive ion mode for
characterizing proteins and protein-metal adducts. To ensure
the best performance and to simulate physiological pH, samples
containing varying drug to protein ratios (from 1:1 to 8:1, protein
concentrations: transferrin, 50 µM; ubiquitin, 25 µM; cyto-
chrome c, 25 µM) were incubated at 37 °C in 20 mM am-
monium bicarbonate buffer adjusted to pH 7.4 by titration with
0.1 M formic acid. Prior to measurement, 30% acetonitrile
(cytochrome c, ubiquitin) or isopropanol (transferrin) and 1%
formic acid (10% in water) were added to the samples to ensure
good spraying conditions; the samples were measured twice after
incubation periods of 30 min and 24 h. The solutions were
introduced via flow injection at a rate of 4 µL/min by using a
Cole-Parmer 74900 single-syringe infusion pump (Vernon Hills,
IL). The ESI-MS instrument was controlled by means of the
Esquire Control software (version 5.2), and all data were
processed using Data Analysis software (version 3.2) (both
Bruker Daltonics).
Cytotoxicity in Cancer Cell Lines. Cell Lines and
Culture Conditions. Human SW480 (colon carcinoma) and
A2780 (ovarian carcinoma) cells were kindly provided by
Brigitte Marian (Institute of Cancer Research, Medical Univer-
sity of Vienna, Austria) and Evelyn Dittrich (General Hospital,
University of Vienna, Austria). Cells were grown in 75 cm2
culture flasks (Iwaki/Asahi Technoglass, Gyouda, Japan) as
adherent monolayer cultures in complete culture medium, i.e.,
Minimal Essential Medium (MEM) supplemented with 10%
heat-inactivated fetal bovine serum, 1 mM sodium pyruvate, 4
mM L-glutamine, and 1% nonessential amino acids (100×) (all
purchased from Gibco/Invitrogen, Paisley, U.K.). The cell lines
5637 and RT-4 (bladder cancer), LCLC-103H and A-427 (lung
cancer), DAN-G (pancreatic cancer), MCF-7 (breast cancer),
KYSE70 (esophagus cancer), and SISO (cervical cancer) were
Acknowledgment. We thank the University of Vienna, the
Hochschuljubila¨umsstiftung Vienna, the Theodor-Ko¨rner-Fonds,
the Austrian Council for Research and Technology Develop-
ment, the FFGsAustrian Research Promotion Agency (Project
FA 526003), the FWFsAustrian Science Fund (Schro¨dinger
Fellowship J2613-N19 [C.G.H.]), the EPFL, and COST D39
for financial support. This research was supported by a Marie
Curie Intra European Fellowship within the 7th European
Community Framework Programme Projects 220890-SuRuCo

924 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 4
Mendoza-Ferri et al.
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Supporting Information Available: Results from elemental
analysis, hydrolysis of 4c, and pKa determination. This material is
available free of charge via the Internet at http://pubs.acs.org.
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